Cross-linking process of carboxylated polysaccharides

ABSTRACT

A process for the preparation of cross-linked polysaccharides containg carboxy groups. The process comprises a first step of activating the carboxy groups in an anhydrous aprotic solvent and then reacting with a polyamine. The cross-linked polysaccharide may be subjected to sulfonation of the five hydroxy groups.

The present invention refers to a cross-linking process of carboxylatedpolysaccharides.

The process of the invention provides a high degree of reproducibilityof the obtained products, in terms of cross-linking degree, homogeneityof the distribution of the cross-linking chains, and chemico-physicalcharacteristics of the products and the technological characteristics ofthe articles obtained therefrom.

The reproducibility is particularly important for the applications inthe medical, pharmaceutical and dermo-cosmetic fields.

The invention further refers to the products obtainable by said processand their applications in the medical, pharmaceutical and dermo-cosmeticfield.

BACKGROUND OF THE INVENTION

The use of macromolecules in the medical/pharrnaceutical field and, morerecently, in the dermatological-cosmetic field, is well established.Macromolecules are used in the preparation of pharmaceuticalformulations as thickening agents, lubricants, gastro-resistant filmcoating agents, particularly in the preparation of capsules, gel,colloids and of different devices (e.g. contact lenses, gauzes, etc.).Macromolecules are also used in the preparation of controlled-releaseformulations of active ingredients.

Reviews of their characteristics and pharmaceutical uses are reported in

1) C. Hansch et Al. Editors—“Comprehensive Medicinal Chemistry”—PergamonPress, Oxford, 1990—Vol. 1-6;

2) A. Wade and P. J. Wellers Editors—“Handbook of PharmaceuticalExcipients”—Ed. 1994—The Pharmaceutical Press.

Said macromolecules belong to different chemical families and may beeither synthetic, or natural or semi-synthetic.

Examples of synthetic macromolecules include polyvinylpyrrolidone,polyoxyethylenealkyl ethers, polyvinyl alcohols, polymethacrylates.Examples of natural macromolecules include native hyaluronic acid (HY)and cellulose.

Examples of semi-synthetic macromolecules includecarboxyalkylcelluloses, widely used in the food and personal careindustries. These macromolecules are characterized by a linear or poorlybranched structure.

A very important modification for increasing the chemical, enzymatic andmechanical strength is provided by cross-linking, which may be carriedout both on synthetic and natural (more or less already modified)polymers.

Examples of cross-linked polymers include polymers used for thegastro-protection of tablets or capsules (polymethacrylates), as well aspolymers used as emulsifiers, suspending agents, tablet hardeners(Carbopol), cross-linked hyaluronic acids, hereinafter discussed.

For the considered applications, and particularly for the preparation ofinvasive medical devices which have to be administered parenterally,said polymers must meet a number of requirements, of technical andregulatory kind.

The technical requirements include:

1) high biocompatibility;

2) resistance to enzymatic systems, both tissular or plasmatic (forinjectable compositions) and gastrointestinal (for oral compositions).

In some cases a gradual degradation, for instance for the controlledrelease of a medicament, may be desirable.

This resistance is particularly important when the macromolecule ispresent in compositions/articles that must last for a long time, e.g.substitutes of the synovial liquid, films, sponges or gels as tissularantiadhesives in different kinds of surgery; in tissular engineering(artificial organs); artificial skins, in the treatment of burns andgenerally in aesthetic surgery;

3) moldability into different shapes (gels, films, sponges, etc.);

4) possibility to be sterilized chemically or physically withoutchanging the product structure.

According to the regulatory requisites, the composition of the differentproduction batches must be kept constant within very narrow limits; thisimplies that the production methods are standardized and that the basecomponents have a very low intrinsic variability.

A possible cause of dishomogeneity for macromolecules derives from thedispersion of molecular weights. Said dishomogeneity becomes even higheras a consequence of cross-linking. This may be a serious drawbackdepending on the field of use and the applicative purposes of the finalproduct.

EP-A-566118 (Kimberly-Clark) discloses cross-linked polysaccharides tobe used as super-absorbents for diapers and similar articles.

The process described therein is based on the cross-linking of celluloseby formation of intermolecular amides, esters or ethers betweenpolyamines, polyols or mixtures thereof and the carboxy group ofpolysaccharides.

The reaction is carried out by heating at about 80° C. the mixture ofthe polysaccharide with the polyol and/or polyamine. This process iscertainly economic and suitable for large scale production where thereproducibility requirements are less stringent.

U.S. Pat. No. 5,465,055 discloses cross-linked polysaccharides(hyaluronic acid and alginic acid) obtained by esterification of COOH ofthe polysaccliaride and OH groups of other molecules, without insertionof cross-linking arms.

WO 91/9119 discloses microcapsules for islets of Langerhans as biohybridorgans, consisting of alginic acid cross-linked with barium ions.

EP 190215 discloses the cross-linking of different polymers(carboxylated starches, dextran, celluloses) with di- or poly-functionalepoxides.

The following cross-linking agents for hyaluronic acids have beenproposed:

polyfunctional epoxides are disclosed in U.S. Pat. Nos. 4,716,224,4,772,419, 4,716,154;

polyalcohols are disclosed in U.S. Pat. No. 4,957,744;

divinylsulfone is disclosed in U.S. Pat. Nos. 4,605,691, 4,636,524;

aldehydes are disclosed in U.S. Pat. Nos. 4,713,448 and 4,582,865;

carboxamides are disclosed in U.S. Pat. No. 5,356,833;

polycarboxylic acids are disclosed in EP-A-718312.

DISCLOSURE OF THE INVENTION

The invention refers to a process for the preparation of cross-linkedpolysaccharides containing carboxy groups, allowing complete control ofcross-linking degree as well as high reproducibility in terms ofconstant characteristics of the final product.

The process of the invention comprises:

a) activation of the carboxy groups of the polysacchatide by reactionwith suitable carboxy activating agents in anhydrous aprotic solvent;

b) reaction of the carboxy activated polysaccharide with a polyamine.

The obtained cross-linked polysaccharide, if desired, may be subjectedto sulphation or hemisuccinylation of the free hydroxy groups.

The products obtainable by the process of the invention may also becomplexed with metal ions such as zinc, copper or iron ions.

The carboxy-containing polysaccharide which may be used according to theinvention may be of natural, synthetic or semi-synthetic origin.Examples of said polysaccharides include Hyaluronic acids (obtained fromtissues or bacteria), carboxymethyldextran, carboxymetbylcellulose,carboxymethyl-starch, alginic acids, cellulosic acid, N-carboxy-methylor butyl glucans or chitosans; heparins with different molecularweights, optionally desulphated and succinylated, derrnatan sulphates,Chondroitin sulphates, heparan sulphates, polyacrylic acids.

Hyaluronic acids, carboxymethylcellulose, heparins, alginic acids andpolyacrylic acids are particularly preferred.

Said cross-linked polymers, obtained by different methods, are known andhave been proposed for several uses (see, for instance, EP 566118,WO91/9119, U.S. Pat. Nos. 5,465,055, EP 190215, EP 718312, U.S. Pat.Nos. 4,716,224 discussed above).

The carboxy activating agents are usually those used in the peptidechemistry: examples of suitable agents include carbonyldiimidazole,carbonyltriazole, chloromethylpyridylium iodide (CMP-J),hydroxybenzotriazole, p-nitrophenol p-nitropheriyltrifluoroacetate,N-hydroxysuccinimide and the like. The use of chloromethylpyridyliumiodide is particularly preferred.

The polyamines have preferably the following general formula:

R₁—NH—A—NH—R₂

wherein R₁ and R₂, which are the same or different, are hydrogen, C₁-C₆alkyl, phenyl or benzyl groups, A is a C₂-C₁₀ alkylene chain, preferablya C₂-C₆ alkylene chain, optionally substituted by hydroxy, carboxy,halogen, alkoxy, amino groups; a polyoxyalkylene chain of formula

[(CH₂)_(n)—O—(CH₂)_(n)]_(m)

wherein n is 2 or 3 and m is an integer from 2 to 10; a C₅-C₇ cycloalkylgroup; an aryl or hetaryl group, preferably 1,3 or 1,4-disubstitutedbenzene. A is preferably C₂-C₆ linear alkylene or a chain of formula

[(CH₂)_(n)—O—(CH₂)_(n)]_(m)

The cross-linking reaction is preferably carried out in a solventselected from tetrahydrofuran, dimethylformamide or dimethyl sulfoxide,and the polysaccharide is preferably salified with a lipophilic cation,for example tetralkylammonium or other lipophilic organic bases.

The transformation of inorganic salts such as sodium salts, intosuitable organic lipophilic salts may be carried out by knownion-exchange methods in homogeneous phase or by precipitation of theacidic component, followed by recovering of the latter and salificationwith the desired organic base.

The activation reaction of the carboxy groups is carried out inhomogeneous phase and in anhydrous polar aprotic solvent.

The polyamine diluted in the same anhydrous solvent, is added to thesolution of the activated ester, keeping the temperature from 0° C. to30° C. The cross-linking reaction times range from 1 to 12 hours, alsodepending on the optional presence of suitable basic substances (e.g.triethylamine).

Generally, the final product is recovered by precipitation of theorganic salt adding a different solvent to the reaction solvent or byevaporation of the latter, followed by centrifugation, washing withdistilled water, repeated dispersions in the solutions of the desiredalkali (for instance sodium, potassium), subsequent washing with waterand final drying of the alkaline salt under vacuum or by lyophilization.

The cross-linking degree (C.L.D) may range within wide limits and may beadjusted by changing the amount of the carboxy activating agents, sincethe activation and the cross-linking reaction are substantiallyquantitative.

The cross-linked polysaccharides obtained according to the invention maybe subjected to sulphation reaction of the hydroxy groups possiblypresent, usually by reaction with the pyridine-sulfur trioxide complexin dimethylformamide.

The reaction is carried out in heterogeneous phase at a temperature of0-10° C. for times ranging from about 0,5 to about 6 hours.

The sulphation degree obtained is comprised within wide limits withrespect to the total of the hydroxy groups and it may be adjusted bychanging the temperature and reaction times. Generally, the sulphationdegree (defined as equivalents of sulphate groups/g) may range from1×10⁻⁶ to 6×10⁻⁶, preferably it is of 2×10⁻⁶ eq/g for a cross-linkingdegree of 0.5.

The cross-linked polymers obtained according to the invention,optionally sulphated, are able to complex metal ions such as zinc,copper or iron ions.

Said complexes may be obtained by dissolving or dispersing untilcomplete swelling the product in water and adding under stirring,preferably at room temperature, a concentrated solution of an organic orinorganic metal salt, e.g. CuCl₂, ZnCl₂, Fe₂(SO₄); after stirring for12-24 hours, the complex is recovered by centrifugation or byprecipitation following the addition of a different solvent (for exampleethanol or acetone) or evaporation under vacuum; the recovered crudeproduct is thoroughly washed with distilled water so as to remove theexcess ions. The complexes are then lyophilized. The content of metalions varies depending on the used operative conditions, particularly thepolymer to ion molar ratios; concentration and pH of the solutions;reaction times and particularly cross-linking degree.

The process of the invention, by suitably adjusting the cross-linkingand/or sulphation degree, allows the preparation of cross-linkedcarboxylated polysaccharides in a wide range of shapes, characterized bydifferent properties such as viscoelasticity, hydration degree,complexing ability towards metal ions, ability to form hydrogels,moldability in films or sponges, mechanical strength of the finalmaterials.

This allows their use in many medical fields, in the human andveterinary field, and in dermo-cosmetology.

The following examples further illustrate the invention.

EXAMPLE 1

Carboxymethylcellulose gel 100% cross-linked with 1,3-diaminopropane.

1,2×10⁻³ moles, with reference to the disaccharide unit of carboxymethylcellulare TBA salt, were dissolved in 30 ml of DMF under N₂ and withstirring. 0.32 g of chloromethylpyridylium iodide (1.2×10⁻³ moles)dissolved in 2 ml of DMF were added dropwise to the solution kept at atemperature of 0° C. with ice.

The molar ratio was 1 to 1 as carboxymethyl cellulose has one functionalcarboxylic group per disaccharide unit. After 20 minutes the solutionwas added with 2 ml of cross-linking 1,3-diaminopropane (0.006 moles),and immediately after also with 0.5 ml of triethylamine. A solid,jelly-like product formed which was washed with DMF, then placed in H₂Oto completely swell.

Alternating washings with EtOH and H₂₀ were then carried out. After thelast washing with EtOH, the product was freeze-dried.

I.R. (film; cm⁻¹): 1650(—CO—NH—); no bending —COO⁻ at 1.400 about.

SD (Swelling Degree, in water and r.t., after 15′; gravimetricdetermination; calculated according to:${{SD} = {\frac{W_{s} - {Wd}}{Wd} \cdot 100}},$

where:

W_(s)=weight of hydrated gel; Wd=weight of dry gel): 7.000

SEM (Scanning Electron Microscopy): the structure looks compact, with15-35 μpers.

The product surface, by rabbit PRP (Platelet Rich Plasma) exposure,shows a very reduced presence of platelets or aggregates in comparisonwith equivalent product obtained by low density polypropylene (ECreference standard).

EXAMPLE 2

Carboxymethyl cellulose gel 50% cross-linked with 1,3-diaminopropane.

1,2×10⁻³ moles, referred to the disaccharide unit of carboxymethylcellulose, were dissolved in 30 ml of DMF under N₂ and with stirring.0.24 g of chloromethylpyridylium iodide (1.2×10⁻³ moles) dissolved in 2ml of DMF were added dropwise to the solution kept at a temperature of0° C. with ice. The molar ratio was 2/1.

After 20 minutes the solution was added with 2 ml of cross-linking1,3-diaminopropane (3×10⁻³ moles), and immediately after also with 0.5ml of triethylamine. A solid, jelly-like product formed which was washedwith DMF, then placed in H₂O to completely swell.

Alternating washings with EtOH and H₂O were then carried out. After thelast washing with EtOH the product was freeze-dried.

I.R. (film; cm⁻¹): 1650(—CO—NH—); no bending —COO⁻ at 1.400 about.

SD: 8.000.

SEM: presence of 13-25 μpers.

Platelet adhesion: as reported in Example 1.

EXAMPLE 3

Alginic acid gel 50% (100% with reference to disaccharide units)cross-linked with 1,3-diaminopropane.

The TBA salt of alginic has been prepared from the sodium salt by ionicexchange on strong cationic resin (Dovex) in H⁺ form (i.e. acidic form),followed by neutralization with tetrabutylammonium hydroxide (TBA—OH)and final liophylisation.

1,2×10⁻³ moles, refered to the monosaccharide unit, were dissolved in 30ml of DMF under N₂ and under stirring. 0.36 g of chloromethylpyridyliumiodide (1,2×10⁻³ moles) dissolved in 2 ml of DMF were added dropwise tothe solution kept at a temperature of 0° C. with ice. The molar ratiowas 1/1.

After 20 minutes the solution was added with 6×10⁻³ moles ofcross-linking 1,3-diaminopropane (0.024 moles), and immediately afteralso with 0.5 ml of triethylamine. A solid, jelly-like product formedwhich was washed with DMF, then placed in H₂O to completely swell.

Alternating washings with EtOH and H₂O were then carried out. After thelast washing with EtOH the product was freeze-dried.

IR (film; cm⁻¹): 1635 (broad) (—CO—NH—): 1.400, about (—COO⁻).

SD: 5.000.

SEM: the structure looks compact and without pores.

EXAMPLE 4

Preparation of hyaluronic acid cross-linked with C.L.D.=0.05 (5% ofavailable carboxy groups). Cross-linking agent: 1,3-propanediamine.

Hyaluronic acid sodium salt (1×10⁻³ mol., with reference to thedisaccharidic unit) were transformed in TBA salt, according to one ofthe following methods:

a) 1% aqueous solution of sodium hyaluronate is transformed in H⁺ formby H⁺ cationic strong resin (Amberlite IR 120); the final solution istreated by a 0,5% solution of TBA—OH to about pH=9.

b) 1% aqueous solution of sodium hyaluronate is transformed in TBA saltsolution by treating with a cationic weak resin in TBA⁺ form. (AmberliteIRC 50)

In both cases, the final solutions are liophylised. The TBA salt is thendissolved in 15 ml of anhydrous DMF, under N₂, and—at 0° C.—0,02 g ofcloromethypyridylium Iodide (CMPJ) in 2 ml of anhydrous DFM, are addeddropwise to the stored solution of TBA salt.

The reaction mixture was then added with 0.1 ml of triethylamine and,then, dropwise, with a solution of 1,3-diaminopropane (d=0.88, in largeexcess, so as to make cross-linking of the activated carboxy groupseasier) in 2 ml of anhydrous DMF. When the addition was over, thereaction mixture was stirred for at least 30′ and the solvent was thenremoved under reduced pressure, the residue was then taken up with DMF,which was subsequently removed by distillation; the residue was thentreated with ethanol, ethanol-water and finally with water.

The product was then lyophilised and the residue subjected to analysis.

IR (film; cm⁻¹): 1630 (—CO—NH); 1740 (—COOH, polysaccharide); 3200(—NH—).

SD (Swelling Degree, in water and r.t., after 15′; gravimetricdetermination; calculated according to:${{SD} = {\frac{W_{s} - {Wd}}{Wd} \cdot 100}},$

where:

W_(s)=weight of hydrated gel; Wd=weight of dry gel): 31.000Cross-linking degree: 0.05 (5% of initially available carboxy groups).

EXAMPLE 5

Preparation of hyaluronic acid cross-linked with C.L.D.=0.05 (5% of theavailable carboxy groups). Cross-linking agent: 1,6-diaminohexane.

Activator: chloromethylpyridylium iodide.

According to the procedure and conditions reported in Example 4, usingthe same HY and the same activator, but 1,6-diaminohexane instead of1,3-diaminopropane, the cross-linked derivative was obtained.

IR (film; cm⁻¹): 1630 (—CO—NH); 1740 (—COOH polysaccharide); 3200(—NH—).

EXAMPLE 6

Preparation of cross-linked hyaluronic acid, with C.L.D.=0.05 (5% of theavailable carboxy groups). Cross-linking agent:0.0′-bis-(2-aminopropyl)PEG500. Activator: chloromethylpyridylium iodideAccording to the procedure and conditions reported in Example 4 andusing the same activator, but 0.0′-bis-(2-aminopropyl)PEG500 instead of1,3-diaminopropane, the cross-linked derivative was obtained. IR (film;cm⁻¹): 1630 (—CO—NH); 1740 (—COOH polysaccharide); 3200 (—NH—).

SD: 31.000.

EXAMPLE 7

Preparation of cross-linked hyaluronic acids, with C.L.D.=0.3 (30% ofthe available carboxy groups). Cross-linking agent: 1,3-propanediamine.Activator: chloromethylpyridylium iodide.

0.6 g of hyaluronic acid tributylammonium salt (1×10⁻³ mol., withreference to the disaccharide unit) were dissolved under stirring in 30ml of DMF under nitrogen. 0.08 g of chloromethylpyridylium iodide(3.5×10⁻⁴ mol) dissolved in 2 ml of DMF were added dropwise to thestirred solution kept at 0° C. The molar ratio was therefore about 3/1.

After 20 minutes 2 ml of 1,3-diaminopropane (0.024 mol) were added,followed immediately by 0.5 ml of triethylamine. A solid, gelatinousproduct was obtained, the product was then swelled with water and washedagain with ethanol.

The final product, after lyophilisation, shows at the scanningmicroscope an irregular pattern with smooth zones alternating to spongyzones.

The cross-linking degree was 0.3 (30% of initially available carboxygroups)

IR (film; cm⁻¹): 1740 (—COOH); 1630 (—CO—NH); 1610 (—COO—); 1560(—CO—NH—).

EXAMPLE 8

Preparation of hyaluronic acid cross-linked with C.L.D.=0.5 (50% of theavailable carboxy groups). Cross-linking agent: 1,3-propanediamine.Activator: chloromethylpyridylium iodide.

0.6 g of hyaluronic acid tributylammonium salt (HY TBA) (1×10⁻³ mol.,with reference to the disaccharide unit) were dissolved under stirringin 30 ml of DMF under nitrogen. 0.15 g of chloromethylpyridylium iodide(CMPJ) (6×10⁻⁶ mol) dissolved in 2 ml of DMF were added dropwise to thesolution, kept at 0° C. The molar ratio was 2HY.TBA:1 CMPJ. After 20minutes, 2 ml of 1,3 diaminopropane (0.024 mol.) were added to thesolution.

0.5 ml of triethylamine were added thereafter.

A solid, gelly-like product was obtained and thoroughly washed with DMF.

After evaporating DMF, the product was swelled in water and washed withethanol before lyophilization.

The obtained product had a cross-linking degree of 0.5 and showed at thescanning microscope a grainy aspect interspaced by large meshes. Athigher magnitudes, the two morphologies appear identical and showround-shaped protrusions a few microns in diameter.

IR (film; cm−1): 1740 (—COOH); 1630 (—CO—NH—); 1610 (—COO—); 1560(—CO—NH—);

The gels were subjected to swelling in PBS and the max swelling abilitywas evaluated.

SD=23.500.

NMR=(13 C; ppm): 29.3 and 39.8 (—CH₂—CH₂—CH₂— propanediamine link);172.5

The rheological properties evaluated on Bohlin VOR Rheometer, at thetemperature of 23=0.1° C., show that the dynamic elastic module G′ (100Pa at 10 Hz) identical at the two considered concentrations (10 and 20mg/ml) is always higher than the viscous dynamic module (G″ 40 Pa for 20mg at 10 Hz and 20 Pa for 10 mg at 10 Hz).

EXAMPLES 9-12

According to the methods disclosed in the previous examples, thecross-linked hyaluronic acid derivatives having the characteristicssummarised in the following table 1, were obtained, starting from 1×10⁻³mol (0.6 g) of hyaluronic acid tributylammonium salt.

The obtained derivatives had the following properties:

TABLE 1 Scanning Cross- Electron Cross-linking agent Amount (g) oflinking Microscopy Ex (mol) CMPJ (mol) degree SD NMR (13) (ppm) I.R.(film) (cm⁻¹) (SEM)  9 1,3-propanediamine 0.6 g (1.210⁻³) (100%)  13.20029.3/39.8 (—CH₂—CH₂—CH₂— 1630 (—CO—NH—); Homogeneouns, (0.024)propanediamine link); 1560 (—CO—NH—); ondulated

morphology. 10 0,0′-1-bis-(-2- 0.15 g (6 × 10⁻⁴) (50%) 9.000 Alternatingdiaminopropyl) smooth areas PEG 500 (0.022) and meshes, circularprotrusions a few microns in size. 11 0,0′-bis (2- 0.15 g (6 × 10⁻⁴)(50%) 6.100 Two aminopropyl)- morphologically PEG 800 (0.022) differentzones, a first one ondulated and a second with hole-like structures. 121,6-diaminohexane 0.15 g (6 × 10⁻⁴) (50%) 8.000 169.46 (—CO—NH— ofcross- 1740 (—COOH); Smooth surface (0.023) linking); 1630 (—CO—NH—);with protrusions 74.04/76.80/83.17/80.41 1610 (—COO); having a few(—CH2— of cross-linking 1560 (—CO—NH—); microns in size. arm)

EXAMPLE 13 Sulphation of 50% Cross-linked HY

The derivative obtained in example 8 was dispersed in 5 nil DMF understrong stirring and nitrogen atmosphere.

A solution of 1 g of SO₃/pyridine in mol of DMF was added at 0° C. andstirred for 3 hours. The reaction was blocked by adding an excess of H₂O(50 ml) and the pH adjusted to 9 with 0.1M NaOH.

The product was thoroughly washed with ethanol and H₂O and thenlyophilized.

The IR spectrum shows, in addition to the bands of the starting product,a peak at 1260 cm⁻¹ and a stronger band at 1025 cm⁻¹.

The gel swells in PBS with SD=33.000. Higher resolution 13C NMR spectrumshows the signals in H₂O at 37° C. reported in table 2. The intensity ofthe NMR signals at 29.3 and 38.8 ppm (—CH₂—) and the signal at 172.5 ppm(CONH) confirm a cross-linking degree of about 50%.

The rheological properties are characterised by dynamic elastic modulesG′ (2500 Pa with 20 mg and 1000 Pa with 10 mg at 10 Hz) which are alwayshigher than the dynamic viscous modules G″ (600 Pa with 20 mg and 150 Pawith 10 mg at 10 Hz) and much higher than the corresponding valuesobtained with non-sulphated HY (13 at 50%—example 5). This compound hasa thrombin time (TT) higher (61±5″) than the control (14.0″) and thecorresponding not cross-linked (14.6″).

The compound was also active in the PRP test using stressed rabbit.

TABLE 2 Table: 13C Chemical shift C-1 C-2 C-3 C-4 C-5 x-C═O y-CH₃ 103.557.3 85.4 71.3 78.7 178.0 25.3 ppm C-1′ C-2′ C-3′ C-4′ C-5′ 6-C═O 105.975.2 76.4 82.8 78.6 176.2 ppm 1-CH2 2-CH2 3-CH2 6′-C═O CROSS-LINKING 39.8 29.3 39.8 172.5 ppm

EXAMPLE 14 Sulphation of Alginic Acid GEL

The cross-linked product after treatment with EtOH was freeze-dried toremove completely humidity and subjected to sulphation of the alcoholgroups.

100 mg of cross-linked product dispersed in 5 ml of DMF were added witha SO₃-pyridine solution of (800 mg in 2 ml of DMF). The reaction shouldbe carried out at 0° C., under nitrogen and with constant stirring for 2hours.

It is mandatory for the product not to adsorb humidity, as it inhibitsthe reaction.

After 2 hours H₂O was added pH was adjusted to 9 by a 1M solution ofNaOH, thereby freeing pyridine.

The thus sulphated product was purified in EtOH.

The analysis of purified products, shows:

IR (film; cm⁻¹) 1263 (stretching SO)

Equivalents of SO₃ groups/g gel (by toluidine complexes):

5% cross linked gel: 6×10⁻⁵

50% cross linked gel: 2×10⁻⁵

100% cross linked gel: 3×10⁻⁵

SD

5% cross linked gel: 19×10³

50% cross linked gel: 9×10⁻³

100% cross linked gel: 7×10⁻³

EXAMPLE 15

Using the same methodology, the sulphated derivatives of 50%cross-linked products according to example 10,11 and 12, have beensynthetized.

Colorimetric characteristics of the sulphated derivatives are reportedin table 3 together with that of the products deriving from examples 8and 13.

TABLE 3 CROSSLINKED POLYMER (50% CROSS-LINKING DEGREE) ΔHa [J/g] Tg [°C.] ΔHb [J/g] Wt % water C.L. Hyal - 1,3 (Ex. 8) 276 51 42 12 C.L.HyalS - 1,3 (Ex. 13) 357 64 53 16 C.L. Hyal - 1,6 (Ex. 12) 327 64 58 16C.L. HyalS - 1,6 465 64 65 20 5 C.L. Hyal - P500.2NH₂ (Ex. 10) 239 45 7210 6 C.L. HyalS - P500.2NH₂ 384 69 113 16 7 C.L. Hyal - P800.2NH₂ (Ex.11) 179 73 30 10 8 C.L. HyalS - P800.2NH₂ 206 76 52 10 Hyal ITBA 164 —130 5 ΔHa [J/g]: water vaporization henthalpy Tg [° C.]: enthalpy forthermal degradation process ΔHb [J/g]: glass transition temperate Wt %water: % of water content, based on ΔHa

EXAMPLE 16

Suphation of carboxymethylcellulose gel.

Following the procedure and conditions reported in Example 14, thesulphated derivative was obtained.

Equivalents of SO₃ groups/g:

a- CMC 5% cross linked: 8×10⁻⁶

b- CMC 50% cross linked: 7×10⁻⁶

c- CMC 100% cross linked: 4×10⁻⁶

SD

a: 20×10³

b: 12×10⁻³

c: 9×10⁻³

What is claimed is:
 1. A process for the preparation of cross-linkedpolysaccharides wherein the cross-linking occurs only through amidebonds between carboxy groups of the starting polysaccharides and aminogroups of a polyamine in which the polysaccharide is selected from thegroup consisting of hyaluronic acids, carboxymethyldextran,carboxymethylcellulose, carboxymethylstarch, alginic acids, celluloseacid, N-carboxy-methyl or butyl glucans or chitosans, heparins withdifferent molecular weights, optionally desulphated and succinylated,dermatan sulphate, chondroitin sulphates and heparan sulphatescomprising (a) activating the caboxy groups of the polysaccharide in anaqueous aprotic solvent using a suitable carboxy activating agent; (b)reacting the carboxy activated polysaccharide with a polyamine selectedfrom the group having the formula R1—NH—A—NH—R2 wherein R1 and R2, whichmay be the same or different, are hydrogen, C 1-C6 alkyl, phenyl orbenzyl groups; A is a C2-C10 alkylene chain; a polyoxyalkylene chain ofthe formula [(CH₂)_(n)—O—CH₂)_(n)]m wherein n is 2 or 3 and m is aninteger from 2 to 10; a C5-C7 cycloalkyl group or an aryl or heteroarylgroup; and (c) recovering the resultant cross-linked polysaccharide. 2.A process according to claim 1 in which the carboxy-containingpolysaccharide is a hyaluronic acid salified with a lipophilic cation;the solvent is selected from tetrahydrofuran, dimethylformamide ordimethyl sulfoxide; the carboxy activating agent ischloromethylpyridylium iodide and the polyamine is one in which A of theformula R₁NH—A-R₂ is a C₂-C₆ linear alkylene chain.
 3. A processaccording to claim 2 in which the polyamine, diluted in a like solventas used in the activation step, is added to the solution of activatedpoly-saccharide to effect the cross-linking reaction in 1-12 hours.
 4. Aprocess according to claim 2 in which the recovered cross-linkedpolysaccharide is sulphated by reaction with a pyridine-sulfur trioxidecomplex.
 5. A process according to claim 2 in which the recoveredcross-linked polysaccharide is complexed with a metal ion selected fromzinc, copper and iron.